The experimental approach of using short, conserved ribosomal primers to generate both conserved rDNA fragments and arbitrary amplification products is presented in U.S. Pat. No. 5,753,467. Microbial identification at the level of genus and species is accomplished by the characterization of variations in length and number of fragments located between highly conserved rDNA sequences. The level of identification is extended to the level of serotype and strain by the concurrent amplification of additional arbitrary regions of the microbial genome. These arbitrary amplification events are referred to as Random Amplified Polymorphic DNA (RAPD).
The advantage of this approach is that the same group of primers are used for all species of microorganism to generate amplification products. Since the sequences of these primers are highly conserved among prokaryotic organisms, these primers are generically applied. A substantial savings of time and expense is realized because the necessity for screening or presumptive identification has been eliminated.
The rDNA genetic locus is a genetic unit, which is found in prokaryotic cells. The conserved amplification targets are those sequences found in the spacer region between the 16S and 23S regions of the rDNA genetic locus. These targets are amplified from conserved sequences in the adjacent 16S and 23S regions. Significant portions of the nucleic acid sequence, which make up this genetic locus, are common to all prokaryotic organisms (FIG. 1 shows a generalized schematic of this locus). The overall relatedness of the 16S, 23S, and 5S regions of this genetic locus has been used as a tool to classify differing species of prokaryotes.
The approach described in U.S. Pat. No. 5,753,467 makes use of short primers of 10-12 bases in length. The products generated by these primers are separated through the use of an electrophoretic separation in either agarose or polyacrylamide. The fragments are then visualized through staining with ethidium bromide. During the gel loading process, the PCR products could potentially contaminate the laboratory environment.